The present invention relates to a modular device for collecting, incubating, and filtering multiple samples.
The use of filter membranes is becoming increasingly fashionable in applications relating to analyzing particles, cells, and bacteria. The calibrated membranes made of polycarbonate produced by firms such as Nucleopore and Millipore have remarkable properties as to their qualities of recovering particles of determined sizes.
They are very fine, they withstand chemical agents, and they lend themselves well to observation under the microscope, either with reflected light or with transmitted light. With transmitted light, it is necessary for the membrane to be transparent in order to make observation possible. With epifluorescent illumination, and providing the cells and bacteria have been stained with a fluorochrcme such as acridine orange, observation shows pale particles on a dark background.
In 1980, Pettipher proposed this method for direct observation of raw milk bacteria for the purposes of quality control. The complex composition of milk and the presence of a high number of cells (several tens of thousands) makes it impossible to filter the samples without prior treatment. The pretreatment consists in treating the milk with a mixture of detergent and trypsin. Under specific conditions of concentration and of timing, the caseine micelles and the fat globules are decomposed and the cells are digested without significantly changing the germ content, thus enabling the germs to be stained.
Filtering is performed on filters of large dimensions using suction. After applying various washes, the stain is applied followed by further washes. This method known as the Direct Epifluorescence Technique (DEFT) presents numerous advantageous. The filtering gives rise to a considerable concentration of events to be counted.
Compared with indirect methods which require information about bacteria to be amplified by proliferation (2 to 3 days), direct methods are supposed to give a quick result. Concentration by filtering can give excellent sensitivity providing the carrier medium is highly filterable The main handicap is the complexity of the method and the sensitivity of its results to preparation and measurement conditions.
Observation is performed under the microscope and when event densities are low, fluctuations in distribution over the filter make it necessary to perform counting in numerous microscope fields. For example, in the application of monitoring raw milk for densities of about 50,000 FCU/ml (FCU =Formant Colony Unit), the DEFT method gives rise to distributions of the order of less than one bacterium per microscope field. In addition, the very poor mechanical properties of the polycarbonate membrane and its fineness make observation under the microscope very difficult.
Placed between slide and cover slip, the combined deformations of the glass slide, of the membrane, and of the microscope stage explain why attempts at performing analysis automatically have been unsuccessful. In spite of the existence of automatic focusing devices, losses of focus are inevitable and a major portion of analysis time is spent in finding focus.
In 1988, Bisconte proposed two solutions which largely satisfy the problems raised and which constitute the subject matter of two patent applications. U.S. Pat. No. 5,190,666, relates to a device making it possible to process several tens of samples automatically and in parallel on the same filter. A second patent application, FR-88 13805, relates to the combination of a means for restraining filters and for placing them on a microscope stage by making use of suction.
The first solution, which integrates the presence of filters mounted in this way in support frames, provides a solution to the question of passing a plurality of samples to be filtered simultaneously, independently, and in parallel, by making use simultaneously of pressure and of suction. In that solution, a receptacle having multiple wells is put into communication with the filter by capillary tubes. The capillaries enable samples and reagents to pass through in succession.
The main drawback of that disposition is to make it difficult to wash the ducting (risk of contamination and stain residues).
The second solution consists mainly in mounting the filters in a semi-rigid support with position indexing. The filter is placed initially in the filtering system in an exact position by means of centering studs. Studs present on the microscope guarantee corresponding positioning during observation. In this way, multiple deposits present on the filter, even if very small in size, can easily be found by a computer program applying X and Y drive to the microscope stage.
The present invention seeks to improve the device for filtering and preparation and to make it simpler and modular, while retaining the same basic concept.